The resolution capability of optical imaging systems is often decisively determined by the object-side aperture of an objective lens and its index of refraction. Light going out from an object can only be detected if it hits the objective within the acceptance angle of the objective. The higher the resolution capability is, the higher the spatial frequencies of the object structure to be imaged which can be detected. The detection of the spatial frequencies is described by the light-optical transfer function or modulation transfer function (in the following: OTF) of the optical systems. The OTF indicates which spatial frequencies, from which the object can be constructed by means of Fourier transformation, are retained in the optical imaging, and/or how parts of the spatial frequencies are attenuated. The resolution capability of the optical system (e.g. a light-optical microscope) is determined by the range in which the OTF of the system does not vanish. If the OTF vanishes completely in sections of reciprocal space, it is impossible, without additional assumptions about the object structure (e.g. spatial limitation, positivity), to reconstruct the corresponding spatial frequencies in an object image. There is general interest in the extension of the OTF in the largest possible region in reciprocal space, in order to increase the resolution of the optical system.
Conventional processes for increasing the resolution capability are particularly directed toward a suitable selection of the object illumination. Thus, for example, in the confocal microscope the object is illuminated on one side preferably point by point with a focused beam of light and simultaneously scanned, with the detection often being limited to one small region of the object by means of a diaphragm (cf. e.g. U.S. Pat. No. 4,631,581).
In the 4Pi microscope described in EP 0 491 289, there is coherent illumination and, depending on the embodiment, also detection on both sides of the object. In the wave field microscope, typically illumination is performed with coherent plane light waves from opposing sides (cf. e.g. U.S. Pat. No. 4,621,911; F. Lanny et al. in “Bioimaging”, Vol. 1, 1993, p. 187 et seq.; U.S. Pat. No. 5,801,881). In the I5M-microscope, there is coherent illumination on both sides and coherent detection, in that the two images of the object are brought into interference on a locally resolving detector (cf. U.S. Pat. No. 5,671,085; M. G. L. Gustafsson et al. in “Proceedings of SPIE”, Vol. 2655, 1996, p. 62 et seq.). A theta microscope is described by E. H. K. Stelzer et al. in “Opt. Commun.”, vol. 111, 1994, p. 536 et seq. and S. Lindeck et al. in “Handbook of Biological Confocal Microscopy”, editor J. B. Pawley, Plenum Press, New York 1995, chapter 26, p. 417 et seq., in which light is detected from three sides, with illumination similar to confocal or 4Pi being used. Because the resolution along the optic axis of the illumination is particularly large for lateral detection in the object plane, one obtains a reduced focused volume overall.
Using a spatially varying (e.g. sinusoidally varying) illumination in stereomicroscopic surface topography processes is also known. By calculating the images measured, conclusions can be drawn about the surface structure of the object (cf. e.g. U.S. Pat. No. 4,525,858; R. Windecker et al. in “Optical Engineering”, vol. 36, 1997, p. 3372 et seq).
A process for high-resolution three-dimensional imaging by detecting optical sections of the object, similarly to confocal microscopy, is described in WO 97/31282. It is based on taking multiple images, each with different patterns, from illumination apertures and associated detection apertures. Through suitable reconstruction processes, an image which is equivalent to that of a confocal microscope can be calculated from the data picked up. This process is also referred to as “Aperture Correlation Microscopy” (cf. also R. Juskaitis et al. in “Nature”, vol. 383, 1996, p. 804 et seq, T. Wilson et al. in “Proceedings of the SPIE”, vol. 2984, 1997, p. 21 et seq). A process is known from WO 98/45745 which is based on illumination with imaging of a diffraction grating or with two interfering laser beams (cf. also T. Wilson et al. in “Cell Vision”, vol. 4, 1997, p. 231 et seq). A similar process for laterally increasing the resolution capability is used in the publication of R. Heintzmann et al. in “Proceedings of SPIE”, vol. 3568, 1999, p. 185 et seq.
The conventional technologies have the following disadvantages. The imaging processes are connected with a relatively large technical outlay. Thus, particularly in the 4Pi, I5M, and theta microscopes, the adjustment is especially difficult. In addition, the processes are difficult to realize because they can be integrated in existing microscopic systems at great expense only. In the wave field microscope, it is a significant problem that the OTF has regions in the axial direction in which it vanishes. In addition, the wave field microscope and/or the 4Pi microscope do not provide any increase in resolution in the lateral direction in comparison with typical epifluorescence microscopy and/or confocal fluorescence microscopy.
Furthermore, many processes (particularly confocal laser scanning, 4Pi, and theta microscopy) are connected with a point by point scanning of the object. This is time-consuming and problematic, above all in the imaging of time-dependent procedures. Scanning processes require very fast detectors (e.g. photomultipliers) which, however, often have a significantly lower detection efficiency than detectors with localized resolution (e.g. CCD's). In fluorescence microscopy, there is the additional problem that the useful illuminance is restricted by the maximum excitation rate of the dye in the focus. This additionally restricts the maximum scanning speed.
Microscopic imaging processes based on non-linear effects are also known. Thus, for example, in U.S. Pat. Nos. 5,034,613, 5,777,732, 5,828,459, and 5,796,112, so-called multiphoton microscopy is described. The confocal effect is achieved in this case by the simultaneous absorption of multiple photons at specific object locations. Other techniques are based on stimulated emission (cf. U.S. Pat. No. 5,731,588, DE-OS 44 16 558) or the depopulation of the ground state of fluorescence molecules by intentionally pumping them into the longer-lived triplet state (cf. S. W. Heil et al. in “Applied Physics B”, vol. 60, 1995, p. 495 et seq).
Until now, no significant increase of the resolution capability has been able to be achieved with the processes based on non-linear optical effects. This is particularly associated with the individual photons having to have relatively low energies and therefore large wavelengths to achieve multiphoton absorptions. In addition, the transfer efficiency at higher spatial frequencies is generally very poor, because typically only a very small part of the illumination pattern contains high spatial frequencies.